Limits...
Genomic Analysis of Storage Protein Deficiency in Genetically Related Lines of Common Bean (Phaseolus vulgaris).

Pandurangan S, Diapari M, Yin F, Munholland S, Perry GE, Chapman BP, Huang S, Sparvoli F, Bollini R, Crosby WL, Pauls KP, Marsolais F - Front Plant Sci (2016)

Bottom Line: SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene.While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter.An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Western Ontario, LondonON, Canada; Genomics and Biotechnology, London Research and Development Centre, Agriculture and Agri-Food Canada, LondonON, Canada.

ABSTRACT
A series of genetically related lines of common bean (Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might also be determined at the translational level.

No MeSH data available.


Related in: MedlinePlus

(A) Sequence reads of lectin locus aligned to the reference BAT-93 genome. Paired end reads from the four genotypes sequenced by Illumina HiSeq2000/2500 were aligned and visualized with IGV. The positions of the lectin genes are indicated by boxes. Gray indicates sequence identity. Color highlights variant bases compared to the reference sequence; Green for A; Red for T; Orange for G; and Blue for C. PDLEC2 and α-amylase inhibitor like protein gene (α-AI-like) are not covered in Sanilac; likewise α-amylase inhibitor 1 (α-AI1) in SARC1 and SMARC1-PN1. BAT-93 and SMARC1N-PN1 have the pdlec1 allele in place of pha-L. (B) Schematic view of polymorphisms in the promoter of α-amylase inhibitor like protein between SMARC1N-PN1 versus SARC1 and SMARC1-PN1 giving rise to unique cis-regulatory elements in SMARC1N-PN1. Analysis of cis-regulatory elements was performed using the Place database. C, CAAACAC element; S, soybean embryo factor 4 binding motif; A, core AACA motif; ABRE, abscisic acid related element.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814446&req=5

Figure 3: (A) Sequence reads of lectin locus aligned to the reference BAT-93 genome. Paired end reads from the four genotypes sequenced by Illumina HiSeq2000/2500 were aligned and visualized with IGV. The positions of the lectin genes are indicated by boxes. Gray indicates sequence identity. Color highlights variant bases compared to the reference sequence; Green for A; Red for T; Orange for G; and Blue for C. PDLEC2 and α-amylase inhibitor like protein gene (α-AI-like) are not covered in Sanilac; likewise α-amylase inhibitor 1 (α-AI1) in SARC1 and SMARC1-PN1. BAT-93 and SMARC1N-PN1 have the pdlec1 allele in place of pha-L. (B) Schematic view of polymorphisms in the promoter of α-amylase inhibitor like protein between SMARC1N-PN1 versus SARC1 and SMARC1-PN1 giving rise to unique cis-regulatory elements in SMARC1N-PN1. Analysis of cis-regulatory elements was performed using the Place database. C, CAAACAC element; S, soybean embryo factor 4 binding motif; A, core AACA motif; ABRE, abscisic acid related element.

Mentions: For analysis of the APA locus, BAT-93, a Mesoamerican genotype, was most similar to SMARC1N-PN1. Figure 3A shows the alignment of the paired end reads to the part of the BAT-93 scaffold00141 containing the APA locus, visualized with IGV. Sequences in gray are identical. Color highlights variant bases. Peak height indicates the number of reads aligned. BAT-93 and other genomic templates were annotated manually after blastn against NCBInr and blastx of individual APA coding sequences against UniProt, based on highest sequence identity to a known lectin accession. In order to annotate the genes in the alignments, reads were joined manually to generate a coding sequence which was used for blastx against UniProt. The gene order was found to be conserved across reference genotypes (BAT-93, G19833, OAC-Rex and the BAC-71F18 from the arcelin-5 genotype). However, the composition of APA genes varied. For the phytohemagglutinin gene located between pha-E and the α-amylase inhibitor like protein gene (Figure 3A), different alleles were found to be present. G19833 and OAC-Rex have pha-L, as do Sanilac, SARC1 and SMARC1-PN1. BAT93 and SMARC1N-PN1 have the pdlec1 pseudogene, previously characterized from Pinto UI111 (Voelker et al., 1986). The presence of the pdlec1 pseudogene in SMARC1N-PN1 and in Great Northern 1140 was confirmed by PCR amplification and sequencing of the PCR products. The sequences isolated were 100% identical to that reported by Voelker et al. (1986). The pdlec1 allele is characterized by a deletion of a single nucleotide, cytosine, after position 32 of the coding sequence, resulting in a premature stop codon at position 132. BAT93 and SMARC1N-PN1 also share the PDLEC2 gene, coding for a leucoagglutinating phytohemagglutinin isoform, further extending the homology with Pinto UI111 (Voelker et al., 1986). G02771, a wild, arcelin-5 genotype, has the arcelin-5 phytohemagglutinin (Kami et al., 2006). Arcelin-5 phytohemagglutinin is 99% identical to pdlec1, but is not a pseudogene. The alignment in Figure 3A confirmed the absence of lec4-B17 and pha-E in SMARC1N-PN1. The alignment also suggested the absence of PDLEC2 and α-amylase inhibitor like protein gene in Sanilac. PDLEC2 and the α-amylase inhibitor 1 gene appeared only partially covered in SARC1 and SMARC1-PN1 suggesting their absence in these genotypes. This conclusion is supported by the Western blotting and genomic PCR data for α-amylase inhibitor 1 (Table 1, Figures 1 and 2). It was not possible to verify the presence of PDLEC2 by genomic PCR due to high degree of sequence identity between leucoagglutinating phytohemagglutinin genes.


Genomic Analysis of Storage Protein Deficiency in Genetically Related Lines of Common Bean (Phaseolus vulgaris).

Pandurangan S, Diapari M, Yin F, Munholland S, Perry GE, Chapman BP, Huang S, Sparvoli F, Bollini R, Crosby WL, Pauls KP, Marsolais F - Front Plant Sci (2016)

(A) Sequence reads of lectin locus aligned to the reference BAT-93 genome. Paired end reads from the four genotypes sequenced by Illumina HiSeq2000/2500 were aligned and visualized with IGV. The positions of the lectin genes are indicated by boxes. Gray indicates sequence identity. Color highlights variant bases compared to the reference sequence; Green for A; Red for T; Orange for G; and Blue for C. PDLEC2 and α-amylase inhibitor like protein gene (α-AI-like) are not covered in Sanilac; likewise α-amylase inhibitor 1 (α-AI1) in SARC1 and SMARC1-PN1. BAT-93 and SMARC1N-PN1 have the pdlec1 allele in place of pha-L. (B) Schematic view of polymorphisms in the promoter of α-amylase inhibitor like protein between SMARC1N-PN1 versus SARC1 and SMARC1-PN1 giving rise to unique cis-regulatory elements in SMARC1N-PN1. Analysis of cis-regulatory elements was performed using the Place database. C, CAAACAC element; S, soybean embryo factor 4 binding motif; A, core AACA motif; ABRE, abscisic acid related element.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814446&req=5

Figure 3: (A) Sequence reads of lectin locus aligned to the reference BAT-93 genome. Paired end reads from the four genotypes sequenced by Illumina HiSeq2000/2500 were aligned and visualized with IGV. The positions of the lectin genes are indicated by boxes. Gray indicates sequence identity. Color highlights variant bases compared to the reference sequence; Green for A; Red for T; Orange for G; and Blue for C. PDLEC2 and α-amylase inhibitor like protein gene (α-AI-like) are not covered in Sanilac; likewise α-amylase inhibitor 1 (α-AI1) in SARC1 and SMARC1-PN1. BAT-93 and SMARC1N-PN1 have the pdlec1 allele in place of pha-L. (B) Schematic view of polymorphisms in the promoter of α-amylase inhibitor like protein between SMARC1N-PN1 versus SARC1 and SMARC1-PN1 giving rise to unique cis-regulatory elements in SMARC1N-PN1. Analysis of cis-regulatory elements was performed using the Place database. C, CAAACAC element; S, soybean embryo factor 4 binding motif; A, core AACA motif; ABRE, abscisic acid related element.
Mentions: For analysis of the APA locus, BAT-93, a Mesoamerican genotype, was most similar to SMARC1N-PN1. Figure 3A shows the alignment of the paired end reads to the part of the BAT-93 scaffold00141 containing the APA locus, visualized with IGV. Sequences in gray are identical. Color highlights variant bases. Peak height indicates the number of reads aligned. BAT-93 and other genomic templates were annotated manually after blastn against NCBInr and blastx of individual APA coding sequences against UniProt, based on highest sequence identity to a known lectin accession. In order to annotate the genes in the alignments, reads were joined manually to generate a coding sequence which was used for blastx against UniProt. The gene order was found to be conserved across reference genotypes (BAT-93, G19833, OAC-Rex and the BAC-71F18 from the arcelin-5 genotype). However, the composition of APA genes varied. For the phytohemagglutinin gene located between pha-E and the α-amylase inhibitor like protein gene (Figure 3A), different alleles were found to be present. G19833 and OAC-Rex have pha-L, as do Sanilac, SARC1 and SMARC1-PN1. BAT93 and SMARC1N-PN1 have the pdlec1 pseudogene, previously characterized from Pinto UI111 (Voelker et al., 1986). The presence of the pdlec1 pseudogene in SMARC1N-PN1 and in Great Northern 1140 was confirmed by PCR amplification and sequencing of the PCR products. The sequences isolated were 100% identical to that reported by Voelker et al. (1986). The pdlec1 allele is characterized by a deletion of a single nucleotide, cytosine, after position 32 of the coding sequence, resulting in a premature stop codon at position 132. BAT93 and SMARC1N-PN1 also share the PDLEC2 gene, coding for a leucoagglutinating phytohemagglutinin isoform, further extending the homology with Pinto UI111 (Voelker et al., 1986). G02771, a wild, arcelin-5 genotype, has the arcelin-5 phytohemagglutinin (Kami et al., 2006). Arcelin-5 phytohemagglutinin is 99% identical to pdlec1, but is not a pseudogene. The alignment in Figure 3A confirmed the absence of lec4-B17 and pha-E in SMARC1N-PN1. The alignment also suggested the absence of PDLEC2 and α-amylase inhibitor like protein gene in Sanilac. PDLEC2 and the α-amylase inhibitor 1 gene appeared only partially covered in SARC1 and SMARC1-PN1 suggesting their absence in these genotypes. This conclusion is supported by the Western blotting and genomic PCR data for α-amylase inhibitor 1 (Table 1, Figures 1 and 2). It was not possible to verify the presence of PDLEC2 by genomic PCR due to high degree of sequence identity between leucoagglutinating phytohemagglutinin genes.

Bottom Line: SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene.While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter.An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Western Ontario, LondonON, Canada; Genomics and Biotechnology, London Research and Development Centre, Agriculture and Agri-Food Canada, LondonON, Canada.

ABSTRACT
A series of genetically related lines of common bean (Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might also be determined at the translational level.

No MeSH data available.


Related in: MedlinePlus